IGEM:MIT/2007/Notebook/2007-8-14

Sequencing of Standard Assembled Parts

• CPX+B0014: Sample 1: okay except single substitution towards end of CPX sequence, Samples 3-4 successful
• R0051+B0034: Samples 1&3 failed to sequence, Samples 2&4 had R0051 only
• I14032+B0034: Samples 1-4 successful

• Next step: 3A ligate I14032+B0034 with CPX+B0014 into pSB1AC3 - use both #4 samples

Double Digestion

8 µl       CPX+B0014 (Sample #4: 135.3 ng/5µl)
0.5 µl     BSA
5 µl       NEB3 Buffer
1 µl       XbaI
1 µl       PstI
34.5 µl    H2O
------------------
50 µl      Total


5 µl       I14032 and B0034 (Sample #4: 225.7 ng/µl)
0.5 µl     BSA
5 µl       NEB2 Buffer
1 µl       EcoRI
1 µl       SpeI
37.5 µl    H2O
------------------
50 µl      Total

Ligations - Take 2

µl   dd pSB1AC3
µl   dd I14032+B0034
µl   dd CPX+B0014
2 µl     10x T4 Ligase Buffer
0.5 µl   T4 Ligase
µl   H20
-------------------
20µl      Total

µl   dd R0051
µl   dd B0014
2 µl     10x T4 Ligase Buffer
0.5 µl   T4 Ligase
µl   H20
-------------------
20µl      Total